Download A combination of renin-angiotensin system polymorphisms is

A combination of renin-angiotensin system polymorphisms is associated with
altered renal sodium handling and hypertension
Alfonso Siania, Paola Russoa, Francesco Paolo Cappucciob, Roberto Iaconec, Antonella
Veneziaa, Ornella Russoc, Gianvincenzo Barbaa, Licia Iacoviellod, Pasquale Strazzulloc
a
b
Epidemiology & Population Genetics, Institute of Food Sciences, CNR, Avellino, Italy;
Department of Community Health Sciences, St. George’s Hospital Medical School,
London, U.K.;
c
Department of Clinical & Experimental Medicine, Unit of Clinical Genetics and
Pharmacology, Hypertension & Mineral Metabolism, "Federico II" University of Naples,
Naples, Italy;
d
“Angela Valenti” Laboratory of Genetic and Environmental Risk Factors for
Thrombotic Disease, Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy .
Short title: RAAS genes, sodium handling and hypertension
Address for correspondence to:
Alfonso Siani, M.D.
Institute of Food Sciences, CNR
Via Roma, 52 A/C
83100 Avellino, Italy
Phone: +39 0825 299353
Fax: +39 0825 299423
e-mail: [email protected]
or to: Pasquale Strazzullo, M.D.
Dept. of Clinical & Experimental Medicine
“Federico II” University of Naples
Via Sergio Pansini, 5
80131 Naples, Italy
Phone: +39 081 7463686
Fax: +39 081 5466152
e-mail: [email protected]
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Methods (expanded version to be published on line)
Study population
The present study used the DNA bank and the database of the Olivetti Heart Study, an
epidemiological investigation of cardiovascular risk factors in men working at the
Olivetti factories in Southern Italy. Between May 1994 and December 1995, 1075 men,
in the age range 25-75 years, were examined (> 95% of the male workforce employed at
the time). The procedures of the study have been described in detail elsewhere (15, 17).
The study protocol was approved by the local Ethics Committee and participants gave
their informed consent to participate. The study included anthropometric and blood
pressure measurements, blood tests and a fixed-sequence questionnaire including
demographic information, past medical history and dietary habits. Specific questions in
the questionnaire were adherence to a low-salt diet and current pharmacological
treatment for hypertension.
Blood pressure and anthropometric measurements
Blood pressure was measured between 8 a.m. and 11 a.m. after the subject had been
sitting upright for at least 10 minutes. Systolic and diastolic (phase V) blood pressure
were taken three times two minutes apart with a random zero sphygmomanometer
(Gelman Hawksley Ltd, Sussex, England). The first reading was discarded and the
average of the second two readings was recorded for systolic and diastolic blood
pressure. Hypertension was defined as a pressure  140 mmHg systolic and/or 90
mmHg diastolic, corresponding to the 80th percentile for our population (or current
antihypertensive drug treatment). The values adopted to define hypertension in our
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population match the 1999 World Health Organization-International Society of
Hypertension cut-off levels for the diagnosis of hypertension (18). Body weight and
height were measured as previously described (15, 17). The body mass index (BMI) was
calculated as weight in kilograms divided by the square of their height in meters.
Renal sodium handling and biochemistry.
The clearance study was carried out in the morning with standardized procedures as
previously described in detail and extensively validated in our laboratory (9, 17, 19-20).
Standard formulae were used to calculate the clearance of creatinine, sodium, lithium and
uric acid and expressed as fractional excretions (%) (9, 17, 19-20). The creatinine
clearance was taken as an index of the glomerular filtration rate and was corrected for
body surface area. The fractional excretion of exogenous lithium could be measured in
677 subjects who accepted to assume the lithium carbonate tablet. No difference in age,
blood pressure, prevalence of hypertension and body mass was found between this group
compared to the remaining study participants. Plasma aldosterone was measured by
radioimmunoassay (DRG Instruments, GmbH-Germany) on 661 participants on their
habitual diet for whom a plasma sample (withdrawn after the participant had been
sitting upright for 10 minutes) was available.
Gene polymorphisms
Genotyping of four renin-angiotensin-aldosterone system polymorphisms was possible
in 918 participants. Polymorphisms I/D of the ACE (21-22), T235C of the
angiotensinogen gene (23), A1166C of the angiotensin II type 1 receptor gene (24)
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and C-344T in the 5’ flanking region of CYP11B2 (25) were typed as previously
described. For each polymorphism a 10% random sample of the study population was
genotyped twice in a blinded fashion with concordant results.
Statistical analysis
Genetic data were analyzed for Hardy-Weinberg equilibrium and for allelic frequency
using the Tools for Population Genetic Analysis version 1.3 (TFPGA available at
http://bioweb.usu.edu/mpmbio/index.htm).
The analysis of the association of genetic variants with phenotypic variables
followed a two- step approach. First, the influence of each candidate gene variant at any
single locus on proximal tubular sodium handling and blood pressure was assessed by
analysis of covariance, accounting for potential confounders, such as age and body mass.
Secondly, to assess the effect of simultaneous variation in different RAAS genes, we
considered all possible combinations of the four variants, thus obtaining eighty-one
genotypes. Out of them, five had empty cells and twenty-eight had fewer than six
individuals in a cell, thus making a comparison of all possible genotype combinations
impracticable despite the relatively large sample size. We then attempted to build a
model based on two assumptions. First, we focused essentially on the homozygous
condition, by assuming that the effect, if any, of multiple variants of candidate genes
would be more evident in the presence of a double copy of the allele . Second, we
selected either the alleles whose association with phenotypes of interest (blood pressure
and/or electrolyte metabolism) has been showed in previous analyses on this population
[that is the C allele of the CYP11B2 (25) and the D allele of the ACE (26)] or the more
frequent alleles in this population (Table 1) ( that is, the M allele of AGT and the A
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allele of AT1R) , in order to obtain groups large enough to allow for sufficient statistical
power. Since only nine subjects carried this complete homozygosity combination,
further analyses were carried out by alternatively adding the heterozygous individuals
at each single locus in separate models. Eventually, we obtained four possible
combinations: a. MM/MT, AA, CC, DD; b. MM, AA/AC, CC, DD;
c. MM, AA, CC/CT, DD; d. MM, AA, CC, DD/ID. All combinations were in turn
compared with the remaining population. In order to reduce the possibility of false
positive results, more stringent criteria were adopted for the statistical evaluation of
results. As four different comparisons were made, type 1 error was set at
0.05/4=0.0125 and 99% CI were used. Fisher’s exact test was used to assess the
independence in 2X2 tables when cells had an expected frequency less than five. The
statistical significance of between-group differences for continuous variables was
assessed by two-sided unpaired T-test. Multiple linear regression models were calculated
to assess whether the allelic combinations obtained according to the model described
above made a statistically significant contribution to the variability of renal tubular
sodium handling. Logistic regression was used to determine the predictive value of
specific allelic combinations with regard to the occurrence of hypertension, accounting
for confounders. To study the attributable risk of hypertension to the presence of the
combination of the four genotype variants, population attributable risk percent (PAR%)
was estimated as follows: PAR%=[(PrevE)(OR-1)]/[(PrevE)(OR-1)+1]x100, where PrevE
is the prevalence of the exposure (frequency of the four genotype variants) and OR is the
estimated odds ratio of the association between the genetic combination and the presence
of hypertension.
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Results were expressed as mean and standard deviation or 99% confidence
intervals (CI), as specified. Statistical analysis was performed using the Statistical
Package for the Social Sciences (SPSS-11.0, Chicago, Illinois, USA).
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